This invention is related to the field of cystic fibrosis. More particularly, it is related to the area of therapeutic treatments and drug discovery for treating cystic fibrosis.
Defects in CFTR, a chloride channel located in the apical membrane of epithelial cells, are associated with the common genetic disease, cystic fibrosis (Quinton, 1986, Welsh and Smith, 1993, Zielenski and Tsui, 1995). CFTR is a 1480 amino acid protein that is a member of the ATP binding cassette (ABC) transporter family (Riordan et al., 1989, Higgins, 1992). Each half of CFTR contains a transmembrane domain and a nucleotide binding fold (NBF), and the two halves are connected by a regulatory, or R domain. The R domain is unique to CFTR and contains several consensus PKA phosphorylation sites (Cheng et al., 1991, Picciotto et al., 1992).
Opening of the CFTR channel is controlled by PKA phosphorylation of serine residues in the R domain (Tabcharani et al., 1991, Bear et al., 1992) and ATP binding and hydrolysis at the NBFs (Anderson et al., 1991, Gunderson and Kopito, 1995). Phosphorylation adds negative charges to the R domain, and introduces global conformational changes reflected by the reduction in the xcex1-helical content of the R domain protein (Dulhanty and Riordan, 1994). Thus, electrostatic and/or allosteric changes mediated by phosphorylation are likely to be responsible for interactions between the R domain and other CFTR domains that regulate channel function (Rich et al., 1993, Gadsby and Naim, 1994).
Rich et al., 1991 showed that deletion of amino acids 708-835 from the R domain (xcex94R-CFTR), which removes most of the PKA consensus sites, renders the CFTR channel PKA independent, but the open probability of xcex94R-CFTR is one-third that of the wild type channel and does not increase upon PKA phosphorylation (Ma et al., 1997, Winter and Welsh, 1997). Thus, it is possible that deletion of the R domain removes both inhibitory and stimulatory effects conferred by the R domain on CFTR chloride channel function. This conclusion is supported by studies that show that addition of exogenous unphosphorylated R domain protein (amino acids 588-858) to wt-CFTR blocks the chloride channel (Ma et al., 1996), suggesting that the unphosphorylated R domain is inhibitory. Conversely, exogenous phosphorylated R domain protein (amino acids 588-855 or 645-834) stimulated the xcex94R-CFTR channel, suggesting that the phosphorylated R domain is stimulatory (Ma et al., 1997, Winter and Welsh, 1997). Therefore, it appears that the manifest activity (stimulatory or inhibitory) depends on the phosphorylation state of the R domain.
About 25% of the known 700 mutations in CFTR produce a mutant CFTR protein which is properly transported to the apical membrane of epithelial cells but have only low level, residual channel activity. There is a need in the art for agents which can boost the level of channel activity in those mutants having low level activity.
It is an object of the present invention to provide an isolated polypeptide useful for enhancing the open probability of CFTR chloride channels.
It is another object of the present invention to provide a method of activating a CFTR protein to enhance its open probability.
These and other objects of the invention are achieved by providing one or more of the embodiments described below. In one embodiment of the invention an isolated polypeptide is provided. The polypeptide comprises a portion of CFTR (cystic fibrosis transmembrane conductance regulator) protein of between 10 and 100 amino acids, said portion comprising 18 amino acids as shown in SEQ ID NO: 1.
In another embodiment of the invention a method is provided for activating a CFTR protein. A polypeptide is applied to a CFTR protein which forms a cAMP regulated chloride channel. The polypeptide consists of a portion of CFTR protein which comprises 18 amino acids as shown in SEQ ID NO: 1, whereby the open probability of the channel formed by the CFTR increases by at least 25%.
According to another aspect of the invention a method is provided for activating a CFTR protein. A polypeptide is applied to a CFTR protein which forms a cAMP regulated chloride channel. The polypeptide consists of a portion of CFTR protein which comprises 22 amino acids as shown in SEQ ID NO: 2, whereby the open probability of the channel formed by the CFTR increases by at least 25%.
The present invention thus provides the art with reagents and tools for enhancing function of channels which are defective in cystic fibrosis patients.